Browsing by Author "Williams, Mark A."

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    Assessment of Exceptional Quality Biosolids for Urban Agriculture
    Alvarez-Campos, Odiney Maria (Virginia Tech, 2019-03-28)
    Biosolids have been used as soil conditioners and fertilizers in agriculture and mine land reclamation, but application of Exceptional Quality (EQ) biosolids to rehabilitate anthropogenic soils for urban agriculture is recent and requires greater study to ensure their appropriate use. The objectives were: 1) to quantify plant available nitrogen (PAN) of new EQ biosolids in a greenhouse bioassay; 2) to quantify PAN of EQ biosolids applied to an urban degraded subsoil via tall fescue N fertilizer equivalency, and compare field results to laboratory tests; 3) to investigate EQ biosolids and inorganic fertilizer effects on urban soil properties, vegetable yields, and potential N and phosphorus (P) loss. Biosolids evaluated were products of thermal hydrolysis plus anaerobic digestion (BLOOM), blending with woody mulch (BM) and sand/sawdust (BSS), composting (LBC), and heat-drying (OCB). Organic N mineralization of new blended biosolids products ranged between 20-25% in the greenhouse bioassay. Products BLOOM, BM, and OCB had the highest organic N mineralization as estimated by the 7-day anaerobic incubation, and this test and soil nitrate-N had the highest correlations with tall fescue N uptake (r=0.49 and r=0.505, respectively). We conducted a two-year field study with four growing seasons (fall 2016-2017 and summer 2017-2018) in an urban disturbed subsoil where EQ biosolids were applied seasonally at agronomic N rates, and yearly at reclamation rates (5x agronomic N). Cabbage yields were greater with reclamation rates (~3.0 kg m-2) and bell pepper yields were greater with BLOOM reclamation rate (~1.0 kg m-2) than with the inorganic fertilizer (1.0 kg m-2 and 0.2 kg m-2, respectively) during second year growing seasons. Soil carbon (C) accumulation (%C remaining in the soil) two years after biosolids additions ranged between 37 to 84%. Soil N availability and mineralization were limited most likely due to lack of residual soil C and N, and high clay content. Nitrogen leaching losses from reclamation rates were not greater than agronomic N rates. Leachate P was below detection during most of the experiment. Despite limiting soil conditions, biosolids amendment at reclamation rates showed greatest potential to increase vegetable yield and improve soil properties after two years of application, while not impairing water quality.
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    Belowground Fungal Community Change Associated with Ecosystem Development
    Pineda Tuiran, Rosana P. (Virginia Tech, 2017)
    Numerous studies have looked at biotic succession at the aboveground level; however, there are no studies describing fungal community change associated with long-term ecosystem development. To understand ecosystem development, the organisms responsible for shaping and driving these systems and their relationships with the vegetation and soil factors, it is critical to provide insight into aboveground and belowground linkages to ultimately include this new information into ecosystem theory. I hypothesized that fungal communities would change with pedogenesis, that these changes would correlate with vegetation community change, and that they should show change of composition and diversity as the seasons change. Chapter 1 discusses the main topics related to this dissertation. Chapter 2 includes a publication draft that describes a study of sand-dune soil samples from northern Michigan that were analyzed to pinpoint the structural change in the fungal community during the development of the ecosystem. The samples were analyzed by pyrosequencing the soil DNA, targeting the internal transcribed spacer region. Chapter 3 contains a coauthored published paper that describes plant invasion of fields in Virginia to determine how they impact soil bacterial and fungal communities. The bacterial and fungal communities that were invaded by 3 different plant species exhibited similar changes, regardless of plant species, suggesting that some functional traits of invasives may have similar impacts on belowground communities. Chapter 4 remarks the conclusions of this research.
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    Changes in belowground biodiversity during ecosystem development
    Delgado-Baquerizo, Manuel; Bardgett, Richard D.; Vitousek, Peter M.; Maestre, Fernando T.; Williams, Mark A.; Eldridge, David J.; Lambers, Hans; Neuhauser, Sigrid; Gallardo, Antonio; Garcia-Velazquez, Laura; Sala, Osvaldo E.; Abades, Sebastian R.; Alfaro, Fernando D.; Berhe, Asmeret Asefaw; Bowker, Matthew A.; Currier, Courtney M.; Cutler, Nick A.; Hart, Stephen C.; Hayes, Patrick E.; Hseu, Zeng-Yei; Kirchmair, Martin; Pena-Ramirez, Victor M.; Perez, Cecilia A.; Reed, Sasha C.; Santos, Fernanda; Siebe, Christina; Sullivan, Benjamin W.; Weber-Grullon, Luis; Fierer, Noah (2019-04-02)
    Belowground organisms play critical roles in maintaining multiple ecosystem processes, including plant productivity, decomposition, and nutrient cycling. Despite their importance, however, we have a limited understanding of how and why belowground biodiversity (bacteria, fungi, protists, and invertebrates) may change as soils develop over centuries to millennia (pedogenesis). Moreover, it is unclear whether belowground biodiversity changes during pedogenesis are similar to the patterns observed for aboveground plant diversity. Here we evaluated the roles of resource availability, nutrient stoichiometry, and soil abiotic factors in driving belowground biodiversity across 16 soil chronosequences (from centuries to millennia) spanning a wide range of globally distributed ecosystem types. Changes in belowground biodiversity during pedogenesis followed two main patterns. In lower-productivity ecosystems (i.e., drier and colder), increases in belowground biodiversity tracked increases in plant cover. In more productive ecosystems (i.e., wetter and warmer), increased acidification during pedogenesis was associated with declines in belowground biodiversity. Changes in the diversity of bacteria, fungi, protists, and invertebrates with pedogenesis were strongly and positively correlated worldwide, highlighting that belowground biodiversity shares similar ecological drivers as soils and ecosystems develop. In general, temporal changes in aboveground plant diversity and belowground biodiversity were not correlated, challenging the common perception that belowground biodiversity should follow similar patterns to those of plant diversity during ecosystem development. Taken together, our findings provide evidence that ecological patterns in belowground biodiversity are predictable across major globally distributed ecosystem types and suggest that shifts in plant cover and soil acidification during ecosystem development are associated with changes in belowground biodiversity over centuries to millennia.
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    Characterization of Novel Type VI Effectors of Acidovorax citrulli and Their Applicability to Biological Control of Plant Diseases
    Wang, Kunru (Virginia Tech, 2022-03-31)
    Bacterial secretion systems have been playing essential roles in modulating the microbiota of most ecological niches. Among a variety of secretion systems, the Type VI Secretion System (T6SS), a nanomachine widely distributed in Gram-negative bacteria, is gaining increasing attention due to its involvement in microbe-microbe and microbe-host interactions through secreting toxins into host cells, microbial competitors, and the extracellular milieu. Most secreted toxins, also known as T6SS effectors, have bacteriostatic effects upon delivery into competing bacteria, and therefore bacteria with potent T6SS may acquire competition advantage and represent promising biological control agents (BCAs). The main body of this dissertation will focus on the characterization of the T6SS of a phytopathogen, Acidovorax citrulli (strain AAC00-1), and the secreted T6 effectors, and will also discuss the possible application of AAC00-1 as a BCA. The seed-borne, gram-negative A. citrulli is able to cause bacterial fruit blotch (BFB) disease and then result in devastating decrease in yields of important cucurbits including watermelon, melon, squash and cucumber. Our inter-microbial competition assays demonstrate that AAC00-1 contains an active T6SS and presents a dramatic antimicrobial activity against a variety of microbes, including Gram-negative bacteria, Gram-positive bacteria, and yeast, dependent upon its T6SS. A group of novel non-enzymatic effectors, Hyde1 proteins, delivered into prey cells through the T6SS, are responsible for this broad-spectrum antimicrobial activity. Expressing Hyde1 or its N-terminal transmembrane domain shows significant toxicity in both E. coli and AAC00-1, and the toxicity of Hyde1 can be counteracted by its immunity protein, Hyde2. A non-pathogenic AAC00-1 strain suppresses the growth of multiple deleterious phytopathogens in planta and protects plant host. Transgenic plants expressing either full-length Hyde1 or its transmembrane domain demonstrate improved resistance against both bacterial and oomycete pathogens. Altogether, we characterize the T6SS killing of AAC00-1, identify the determinant effectors and discuss the application of both AAC00-1 and its T6SS effector in plant disease management. Additionally, in order to develop molecular tools better serving our T6SS-related studies, we successfully generate a series of salicylic acid (SA)-inducible vectors, functioning in A. citrulli, that can be used for inducible gene expression, protein purification and other applications. The core regulatory component that we employ, is a transcriptional regulator, Sal7AR-V295F, due to its responsiveness to salicylate. By cloning this fragment to a broad-host-range plasmid, in this study, we establish multiple SA-inducible vectors that may be used in most Gram-negative bacteria. When using the E. coli strain C41(DE3) as the expression host, protein purification can be conducted routinely, upon the addition of affinity tags to our vectors, such as the maltose-binding protein (MBP) tag. Combining the modified vectors with the robust NanoLuc binary Technology (NanoBiT), we are able to devise a novel bacteria two-hybrid system as an effective method to detect protein-protein interaction. Two complementary fragments of the NanoLuc protein, LgBiT and SmBiT, with extremely low affinity, are fused to potential interactors, and they will be brought into proximity and reconstitute NanoLuc bioluminescence upon the occurrence of interaction. This system is used in our T6SS study to validate the interaction between Hyde1 toxin and its cognate immunity protein. Another fragment, HiBiT, which automatically interacts with LgBiT and reconstitutes NanoLuc, is cloned to the SA-inducible vector as well, enabling us to generate a split-NanoLuc-based method, for the purpose of detecting secretion of tagged T6 toxins into the prey bacterial cells expressing LgBiT. Overall, our SA-inducible vectors and their further modifications enrich the molecular tool repertoire for T6SS-related studies.
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    Compost applications increase bacterial community diversity in the apple rhizosphere
    Sharaf, Hazem; Thompson, Ashley A.; Williams, Mark A.; Peck, Gregory M. (2021-03-24)
    Sustainable practices are key to the improvement of soil fertility and quality in apple (Malus x domestica Borkh.) orchards. Rootstock genotype and fertilizer inputs can alter soil biology, as well as aboveground traits including nutrient acquisition. In this study, a factorial design was used to assess the interaction between two apple rootstocks, 'Geneva 41' ('G.41') and 'Malling 9' ('M.9') with four fertilizer treatments [chicken-litter compost, yardwaste compost, fertigation using Ca(NO3)(2), and an unamended control]. The bacterial community in the rhizosphere was assessed for its impact on both plant and soil properties for each rootstock x fertilizer treatment combination. The bacterial community was dominated by Acidobacteria, Proteobacteria, and Planctomycetes, but Verrucomicrobia and Chloroflexi were the most responsive to the fertilizer treatments. The chicken litter and yardwaste treatments had a greater effect on bacterial community structure than the control. Yardwaste, in particular, was associated with increased relative abundance of Chloroflexi, which was correlated with soil nutrient concentrations. Malling 9 had a greater bacterial diversity than G.41, but the rootstock treatment had no independent effect on the rhizosphere community structure. There was, however, a strong interaction between the rootstock and fertilizer treatments. Carbon cycling was the most prominent functional change associated with the soil bacterial community. These results suggest that compost amendments have a more positive effect on soil bacterial activity and nutrient availability than Ca(NO3)(2). Our work shows that waste-stream amendments can lead to multiple positive responses, such as increasing aboveground tree biomass, thus potentially improving orchard productivity.
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    Conservation agriculture in urban deserts
    Edralin, Don Immanuel A.; Hok, Lyda; LeNgoc, K.; Williams, Mark A.; Gayle, G.; Raczkowski, Charles W.; Reyes, Manuel R. (2012)
    Limited access to nutritious and affordable food is experienced by 23 million people in the US as they live in 'food desserts' making them food and health insecure. Resources such as land, water, labor and capital are used not in the context of sustainability making the problem more severe. Urban conservation agriculture will be an ‘oasis’ or a sustainable solution to this problem on food desserts and unsustainable resource use. A part of a human disturbed landscape, a turf grass lawn, was converted into ‘oasis sofas’, a 3’ by 6’ vegetable production area outlined by wood, following conservation agriculture principles of minimum soil disturbance, continuous mulch and diverse species at North Carolina Agricultural and Technical State University. Rainwater was used for irrigation and leguminous cover crops used to increase soil fertility. The cost of maintaining oasis sofas’ were seen to be lower than maintaining an equal amount of turf lawn. Oasis sofas’ adds beauty and diversity to the lawn while it gives nutritious food to the household. Fall yield of unfertilized vegetables; broccoli, collard greens, kale and lettuce were 4.5, 2.8, 1.7 and 2.6 kilograms, respectively, per ‘oasis sofa’. Part of the capital and hired labor to maintain turf grass lawns may be used to maintain oasis sofa’s which would lead to greater benefits as it brings nutritious food to the household. Oasis sofas ease access to homegrown healthy food which would likely improve the household’s food and health security.
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    COREMIC: a web-tool to search for a niche associated CORE MICrobiome
    Rodrigues, Richard R.; Rodgers, Nyle C.; Wu, Xiaowei; Williams, Mark A. (PeerJ, 2018-02-15)
    Microbial diversity on earth is extraordinary, and soils alone harbor thousands of species per, gram of soil. Understanding how this diversity is sorted and selected into habitat niches is a major focus of ecology and biotechnology but remains only vaguely understood. A systems-biology approach was used to mine information from databases to show how it can be used to answer questions related to the core microbiome of habitat-microbe relationships. By making use of the burgeoning growth of information from databases, our tool "COREMIC" meets a great need in the search for understanding niche partitioning and habitat-function relationships. The work is unique, furthermore because it provides a user-friendly statistically robust web-tool (hit p://cot eiruc2.appspot.corn or http://corc-mic.com), developed using Google App Engine, to help in the process of database mining to identify the "core microbiome" associated with a given habitat. A case study is presented using data from 31 switchgrass rhizosphere community habitats across a diverse set of soil and sampling environments. The methodology utilizes an outgroup of 28 non-switchgrass (other grasses and forbs) to identify a core switchgrass microbiome. Even across a diverse set of soils (five environments), and conservative statistical criteria (presence in more than 90% samples and FDR q-val <0.05% for Fisher's exact test) a core set of bacteria associated with switchgrass was observed. These included, among others, closely related taxa from Lysobacter spp., Mesorhizobium spp, and Chitinophagaceae. These bacteria have been shown to have functions related to the production of bacterial and fungal antibiotics and plant growth promotion. COREMIC can be used as a hypothesis generating or confirmatory tool that shows great potential for identifying taxa that may be irnportant to the functioning of a habitat (e.g. host plant). The case study, in conclusion, shows that COREMIC can identify key habitat-specific microbes across diverse samples, using currently available databases and a unique freely available software.
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    Development of Ecosystem Structure and Function on Reforested Surface-Mined Lands
    Avera, Bethany Noel (Virginia Tech, 2015-01-30)
    Surface mining in the central Appalachian coalfield disturbs landscapes. Post-mining reforestation efforts now achieve successful reestablishment and growth; however, it is unclear whether reforestation efforts also restore the native forest ecosystem functions. We quantified rates of return of key ecosystem functions and structural attributes of the post-mining forested ecosystem. A chronosequence of four reforested mine sites and an unmined reference stand were studied in southwestern Virginia. Total soil nitrogen (N) and component (mineral soil, forest floor, root, and aboveground biomass) ecosystem carbon (C) pools were quantified. Throughout the growing season, soil gas fluxes [i.e., carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4)], soil inorganic-N [nitrate (NO3-) and ammonium (NH4+)], and total and active microbial biomass were measured. Soil organic C (SOC) and total ecosystem C are returning to the mined landscape. Ecosystem C was correlated with N (r= 0.80; p= 0.0003) and with total and active microbial biomass (r= 0.92; p=<.0001 and r= 0.86; p=<.0001). Available soil inorganic-N and CO2 and N2O fluxes showed no significant differences among study sites; however, the reforested mine soils showed a diminished capacity for CH4 uptake. Although some ecosystem components and functions rapidly returned to the mined landscape, others did not. Our results indicate that reforestation on surface mined lands is largely successful at restoring many ecosystem functions, yet certain functions are decoupled from the redeveloping ecosystem structure. Improved understanding of relationships between ecosystem functions and structural measures in this context can aid development of ecosystem restoration science and mine reclamation practice.
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    Do Roots Bind Soil? Comparing the Physical and Biological Role of Plant Roots in Streambank Fluvial Erosion
    Smith, Daniel Jeremy (Virginia Tech, 2022-09-22)
    This study is the first to consider how the combination of root physical effects, microbial production of EPS, and root effects on the hydrodynamic boundary layer could influence streambank soil erodibility. Specifically, the goal of this research was to quantify the physical and biological effects of roots on streambank fluvial erosion. A series of laboratory-scale erosion tests were conducted using a mini jet erosion testing device and a recirculating flume channel to address this goal. Several soil and vegetation factors that influence fluvial entrainment, like extracellular polymeric substances (EPS), soil aggregate stability and root length density, were measured following erosion testing. For flume experiments, three streambank boundary conditions were constructed to simulate unvegetated streambanks, as well as streambanks with herbaceous and woody roots. Soil treatments were also created to represent unamended and organic matter (OM) amended soil either without roots (bare soil), with synthetic roots, or with living roots (Panicum virgatum). Median soil erosion rates along the simulated rooted boundaries were two to ten times higher compared to the unvegetated boundary due to protruding root impacts on the boundary layer. In flume experiments, median erosion rates were 30% to 72% lower for unamended soils containing compacted synthetic root fibers as compared to bare soil samples. Adding both OM and fibers to the soil had a greater effect; the median erosion rate reductions of live rooted treatments (95% to 100%) and synthetic rooted + OM treatments (86% to 100%) were similar and statistically lower than bare soil controls. Stimulated microbial production of EPS proteins were significantly correlated with increased erosion resistance in OM-amended treatments while OM treatments had significantly lower EPS carbohydrates compared to unamended treatments. In summary, while sparsely spaced roots exposed on streambanks may increase soil erosion rates due to impacts on the hydrodynamic boundary layer, overall results highlight how the synergistic relationship between root fibers and soil microbes can significantly reduce streambank soil erodibility due to fiber reinforcement and EPS production.
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    Drought-induced soil microbial amino acid and polysaccharide change and their implications for C-N cycles in a climate change world
    Kakumanu, Madhavi L.; Ma, Li; Williams, Mark A. (Springer Nature, 2019-07-29)
    High microbial carbon (MBC) demand, a proxy for energy demand (cost), during soil microbial response to stressors such as drought are a major gap in understanding global biogeochemical cycling of carbon (C) and nitrogen (N). The dynamics of two dominant microbial pools (amino acids; AA and exopolymeric substances; EPS) in soils exposed to drying and C and N amendment to mimic both low and high nutrient soil habitats were examined. It was hypothesized that dynamics of EPS and AA (osmolytes) would be greater when soil drying was preceded by a pulse of bioavailable C and N. Drying reduced AA content, even as overall soil MBC increased (similar to 35%). The increase in absolute amounts and mol% of certain AA (eg: Taurine, glutamine, tyrosine, phenylalanine) in the driest treatment (-10 MPa) were similar in both soils regardless of amendment suggesting a common mechanism underlying the energy intensive acclimation across soils. MBC and EPS, both increased similar to 1.5X and similar to 3X due to drying and especially drying associated with amendment. Overall major pools of C and N based microbial metabolites are dynamic to drying (drought), and thus have implications for earth's biogeochemical fluxes of C and N, perhaps costing 4-7% of forest fixed photosynthetic C input during a single drying (drought) period.
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    Ecology of Root Nodule Bacterial Diversity: Implications for Soybean Growth
    Sharaf, Hazem (Virginia Tech, 2021-11-30)
    Diazotrophs supply legumes such as soybean (Glycine max L. Merr) with nitrogen (N) needed for protein synthesis through biological nitrogen fixation (BNF). Through BNF, these bacteria such as Bradyrhizobium that reside in soybean root nodules, convert atmospheric nitrogen (N2) into ammonia (NH3/ NH4), a form that is biologically available for use by the plants, in return for photosynthate carbon from the plant. Abiotic stresses such as drought disrupt BNF and subsequently affects soybean yield. In addition, increasing demand for soybean is leading to supplementing its growth with synthetic N fertilizer. However, fertilizer application is known for its detrimental effects on the environment causing waterways eutrophication contributing to global warming. On the other hand, diazotrophs can supply soybean with up to 90% of N need. As such, improving the understanding and exploiting the relationship between soybean and diazotrophs is key to promoting the sustainable growing of soybean. This dissertation here investigates three main questions. First, how the soybean-diazotrophs respond to changes in water such as rainfall and irrigation. Second, how changes in these bacterial diazotrophs are related to levels of BNF, and N-related soybean molecular markers. Finally, as my colleagues and I found non-diazotrophs in the nodules of some soybean plants, I was curious about the role they are playing inside the nodules in concert with the diazotrophs. The main hypotheses tested in this dissertation are that root nodule bacterial community (bacteriome) would (1) vary by plant type, (2) respond to changes in water, and (3) be related to BNF. To answer the research questions, I devised the dissertation as follows. In Chapter 2, my colleagues and I used nine commercial cultivars of soybean that vary in drought tolerance and agronomic traits. We show that soybean sometimes, but not always, harbor a consortium of non-nitrogen fixing bacteria belonging to Pseudomonadaceae and Enterobacteriaceae families. However, as expected, nodules diazotrophs rather than non-diazotrophs responded most to changes in soil water status. In chapter 3, I used a collection of 24 genotypes of soybean that vary in their ability to fix nitrogen. The results revealed that the bacteriome diazotroph alpha diversity metrics, phylogenetic richness and evenness, was correlated with changes in BNF. Moreover, few N-related molecular markers were associated with some of the bacteria. However, we have also observed a strong effect of the environment on the diazotroph driven process of BNF (i.e. 39%-75%). For chapter 4, we sequenced three of the Pseudomonas spp. strains that were subsequently recovered again from a diversity of soybean nodules in field trials. I found that one of the strains has the ability to adapt to the nodule's unique hypoxic conditions, supporting Bradyrhizobium nodulation and possibly nodule iron. The results include the draft assembly of the proposed Pseudomonas nodulensis sp. nov. as a novel species of nodule adapted bacteria belonging to the P. fluorescens complex. The results of this dissertation contribute to the basic knowledge needed to advance sustainable breeding and management of soybean. Nodule diazotrophs are sensitive to water status e.g. drought, and other experiments have shown that the nodule bacteriome is the driver of BNF. Thus, improving the understanding and exploiting the nodule bacteriome will support developing more resilient cultivars of soybean that are efficient in BNF, and tolerant of stress. Identifying and testing diazotrophs and atypical nodule bacteria will provide a platform for developing new inoculants and biofertilizers.
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    Effects of Fertilizer Source and Rate on Zinnia Cut Flower Production in a High Tunnel
    Bi, Guihong; Li, Tongyin; Gu, Mengmeng; Evans, William B.; Williams, Mark A. (MDPI, 2021-09-23)
    Sustainable nutrient management in high tunnel production is critical for optimizing crop yield and quality and improving soil health. In this study, we investigated the influence of different pre-plant composts (composted broiler litter, vemicompost, and cotton gin compost) in combination with different rates of organic or conventional fertilizer on zinnia plant growth, marketable yield of cut flower stems (>30 cm), and soil nutrients in a high tunnel over two years. Results showed that in general, pre-plant compost influenced plant growth, and plants that received composted broiler litter had the highest plant growth index. However, pre-plant compost did not affect the number of marketable cut stems. Fertigation during the growing season influenced the number of marketable cut stems. Comparable rates of nitrogen, from either organic or conventional fertilizer, produced similar numbers of marketable stems, suggesting that the organic fertilizer used in this study can be used as a fertilizer source for the production of zinnia cut flowers. After two years of production under the high tunnel, soil-extractable phosphorus, sodium, zinc, and pH significantly increased, suggesting that salt accumulation should be closely monitored in response to different compost or fertilizer sources with long-term production under high tunnels.
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    The Effects of Rootstock Selection and Carbon-based Fertility Amendments on Apple Orchard Productivity and Soil Community Ecology
    Thompson, Ashley A. (Virginia Tech, 2016-12-08)
    In apple (Malus domestica Borkh.) orchards, rootstock genotype, and soil fertility management practices impact soil fertility, plant associated soil microbial communities, and orchard productivity. Apple growers select rootstocks to confer beneficial traits, including size control, precocity, and pest and disease resistance. Rootstock genotype may also influence microbial communities, resulting in changes that affect tree health and productivity. Many apple growers apply synthetic nitrogen fertilizers to improve fruit yield and quality. In excess of tree requirements, nitrogen fertilizers may reduce crop yield and quality, as well as contribute to water pollution. The addition of carbon-based amendments, such as yardwaste, chicken litter composts, and biochar, may potentially reduce nitrogen and water loss, while improving soil structure and mineral nutrient availability. Orchard and pot-in-pot experiments were designed to study the following objectives: 1) determine the effects of integrated carbon-based fertilizer amendments on tree growth, productivity, and orchard soil fertility, 2) assess the effects of biochar on tree growth, leaf mineral nutrition, soil physiochemistry, and microbial community structure and activity, and 3) understand how rootstocks and fertilizers alter soil microbial communities. Applications of composts, integrated compost-calcium nitrate fertilizers, and biochar increased soil carbon, organic matter, cation exchange capacity and microbial respiration. In the orchard study, nitrogen fertilizer application did not increase tree growth, fruit quality, or leaf nitrogen concentration. Biochar applied at high rates with nitrogen fertigation increased tree growth and leaf nitrogen concentration similar to nitrogen fertigation. In the pot-in-pot compost study, chicken litter compost increased tree growth, and integrated compost-calcium nitrate fertilizer applications increased leaf N concentration. Analysis of the microbial community structure of bulk soil samples from the biochar and compost pot-in-pot experiments determined that the community structure was similar for all treatments during the three-year study. Metagenomic sequencing of the rhizosphere bacterial community indicated that compost applications altered community diversity and evenness, and that compost treatments were more similar to each other than to the calcium nitrate treatment. Data from my dissertation research suggests that compost can be used to increase orchard soil fertility, tree growth, and leaf nutrition, and that compost applications increase soil microbial community diversity and activity.
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    Elucidation of the Specificity of S. meliloti Chemoreceptors for Host Derived Attractants
    Webb, Benjamin A. (Virginia Tech, 2016-08-24)
    The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields.
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    Exploring candidate genes and rhizosphere microbiome in relation to iron cycling in Andean potatoes
    Xiao, Hua (Virginia Tech, 2017-06-05)
    Fe biofortification of potato is a promising strategy to prevent Fe deficiency worldwide either through traditional breeding or biotechnological approaches. These approaches require the identification of candidate genes to uptake, transport and store Fe in potato tubers. We employed multiple approaches including SNP genotyping, QTL analysis, identifying genes orthologous to Arabidopsis ferrome, yeast complementation assay and genetic transformation to avoid the limitation from a single approach. We revealed several candidate genes potentially associated with potato plant Fe acquisition, PGSC0003DMG400024976 (metal transporter), PGSC0003DMG400013297 (oligopeptide transporter), PGSC0003DMG400021155 (IRT1) and IRTunannotated (an ortholog to the IRT gene that is not annotated in the potato genome). The microorganisms in the rhizosphere react intensely with the various metabolites released by plant roots in a variety of ways such as positive, negative, and neutral. These interactions can influence the uptake and transport of micronutrients in the plant roots. Therefore, the contribution of soil microorganisms in the rhizosphere to improve Fe supply of plants may play a key role in Fe biofortification, especially under real world field-based soil scenarios. We thus investigated rhizosphere microbial community diversity in Andean potato landraces to understand the role of plant-microbial interaction in potato Fe nutrient cycling. From the analysis of the high-throughput Illumina sequences of 16S and ITS region of ribosomal RNA gene, we found that both potato landraces with low and high Fe content in tubers and a landrace on which low or high Fe content fertilizer was applied to the leaf surface had large impacts on the rhizosphere fungal community composition. Indicator species analysis (ISA) indicated that Operational Taxonomic Units (OTUs) contributing most to these impacts were closely related to Eurotiomycetes and Leotiomycetes in the phylum Ascomycota, Glomeromycetes in the phylum Glomeromycota and Microbotryomycetes in the phylum Basidiomycota. Lots of species from these groups have been shown to regulate plant mineral nutrient cycling. Our research revealed potential candidate genes and fungal taxa involved in the potato plant Fe nutrient dynamics, which provides new insights into crop management and breeding strategies for sustainable Fe fortification in agricultural production.
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    Foliar application of Fe resonates to the belowground rhizosphere microbiome in Andean landrace potatoes
    Xiao, Hua; Rodrigues, Richard R.; Bonierbale, Merideth; Veilleux, Richard E.; Williams, Mark A. (2018-10)
    Iron (Fe) is a crucial nutrient for plant growth (e.g. chlorophyll production), and though it is one of the most abundant elements in soil, very low bioavailability can limit plant growth. Studies indicate that many soil bacteria and fungi (e.g. mycorrhizal) play a role in Fe nutrient cycling and plant production, but the evidence for fungal support of plant growth is overwhelmingly correlative and in need of experimental corroboration. An Andean native potato landrace was grown in a greenhouse under Fe limitation and using three levels (Low, Medium, High) of foliar fertilization (FeEDDHA). Application occurred at 45, 60 and 70 days of growth corresponding to periods where Fe limitation is expected to be greatest. The rhizosphere soils were sampled at the flowering stage (80 days). Soil bacterial and fungal communities were examined using high-throughput sequencing of 16S and ITS regions of ribosomal RNA gene, respectively, followed by analysis using Quantitative Insights Into Microbial Ecology (QIIME v1.8). Multivariate data analyses showed that Fe fertilization of leaves significantly (p < 0.05) influenced the beta diversity of fungi but not bacterial communities in the rhizosphere. Using our novel approach, it was expected and confirmed that fungal communities would shift and mycorrhizal genera (Glomus) would be altered, however, the degree to which community change was observed was more than expected. Glomeromycota (-16.3%) related to the family Gigasporaceae accounted for 2.8% of OTU and were 2-3 times greater in the rhizosphere of high relative to medium and low Fe conditions. Overall, the results indicate that foliar addition of Fe influences plant Fe and resonates into the root system to affect rhizosphere fungal communities. Potato Fe status thus appears to impact potato root-fungal interactions potentially mediated through mycorrhizal fungi.
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    Global ecological predictors of the soil priming effect
    Bastida, Felipe; Garcia, Carlos; Fierer, Noah; Eldridge, David J.; Bowker, Matthew A.; Abades, Sebastian R.; Alfaro, Fernando D.; Berhe, Asmeret Asefaw; Cutler, Nick A.; Gallardo, Antonio; Garcia-Velazquez, Laura; Hart, Stephen C.; Hayes, Patrick E.; Hernández, Teresa; Hseu, Zeng-Yei; Jehmlich, Nico; Kirchmair, Martin; Lambers, Hans; Neuhauser, Sigrid; Pena-Ramirez, Victor M.; Perez, Cecilia A.; Reed, Sasha C.; Santos, Fernanda; Siebe, Christina; Sullivan, Benjamin W.; Trivedi, Pankaj; Vera, Alfonso; Williams, Mark A.; Moreno, Jose Luis; Delgado-Baquerizo, Manuel (Springer Nature, 2019-08-02)
    Identifying the global drivers of soil priming is essential to understanding C cycling in terrestrial ecosystems. We conducted a survey of soils across 86 globally-distributed locations, spanning a wide range of climates, biotic communities, and soil conditions, and evaluated the apparent soil priming effect using C-13-glucose labeling. Here we show that the magnitude of the positive apparent priming effect (increase in CO2 release through accelerated microbial biomass turnover) was negatively associated with SOC content and microbial respiration rates. Our statistical modeling suggests that apparent priming effects tend to be negative in more mesic sites associated with higher SOC contents. In contrast, a single-input of labile C causes positive apparent priming effects in more arid locations with low SOC contents. Our results provide solid evidence that SOC content plays a critical role in regulating apparent priming effects, with important implications for the improvement of C cycling models under global change scenarios.
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    Identification and characterization of ice nucleation active bacteria isolated from precipitation
    Failor, Kevin Christopher (Virginia Tech, 2018-02-05)
    Since the 1970s, a growing body of research has suggested that bacteria play an active role in precipitation. These bacteria are capable of catalyzing the formation of ice at relatively warm temperatures utilizing a specific protein family which aids in the binding of water molecules. However, the overall biodiversity, concentration, and relationship of ice nucleation active (ice+) bacteria with air mass trajectories and precipitation chemistry is not well studied. Precipitation events were collected over 15 months in Blacksburg, VA and ice+ bacteria were isolated from these samples. From these samples, 33,134 total isolates were screened for ice nucleation activity (INA) at -8 °C. A total of 593 of these isolated positively confirmed for INA at the same temperature in subsequent tests. The precipitation events had a mean concentration of 384±147 colony forming units per liter. While the majority of confirmed ice+ bacteria belonged to the gammaproteobacteria, a well-studied class of bacteria, including ice+ species of Pseudomonas, Pantoea, and Xanthomonas, two isolates were identified as Lysinibacillus, a Gram-positive member of the Firmicute phylum. These two isolates represent the first confirmed non-gammaproteobacteria with INA. After further characterization, the two isolates of Lysinibacillus did not appear to use a protein to freeze water. Instead, the Lysinibacillus isolates used a secreted, nanometer-sized molecule that is heat, lysozyme, and proteinase resistant. In an attempt to identify the mechanism responsible for this activity, species type strains were tested for INA and UV mutants were generated to knock out the ice+ phenotype. Based on these results, only members of the species L. parviboronicapiens exhibit INA and the genes responsible for the activity may lie within a type-1 polyketide synthase/non-ribosomal peptide synthase gene cluster. This gene cluster is absent from the genomes of all non-ice+ strains of Lysinibacillus, and contains mutations in five of the nine ice nucleation inactive mutants generated from the rain isolated strain. To better understand the phylogenetic relationship among ice+ Lysinibacillus, a comprehensive reference guide was compiled to provide the most up-to-date information regarding the genus and each of its species. This reference will be available to other researchers investigating Lysinibacillus species or other closely related genera.
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    Identification, Characterization, and Use of Precipitation-borne and Plant-associated Bacteria
    Mechan Llontop, Marco Enrique (Virginia Tech, 2020-01-10)
    Bacteria are ubiquitously present in every ecosystem on earth. While bacterial communities that reside in specific habitats, called the microbiota, have characteristic compositions, their constituents are exchanged between habitats. To understand the assembly processes and function of a microbial community in an ecosystem, it is thus important to identify its putative sources and sinks. The sources and sinks of the plant leaf microbiome, also called the phyllosphere microbiome, are still under debate. Here, I hypothesized that precipitation is a so far neglected source of the phyllosphere microbiome. Using 16S rRNA amplicon and metagenomic sequencing, I identified the genera Massilia, Sphingomonas, Methylobacterium, Pseudomonas, Acidiphilium, and Pantoea as members of the core rain microbiome in Blacksburg, VA. Further, I used rainwater as a bacterial inoculum to treat tomato plants. I showed that rain-borne bacteria of the genera Chryseobacterium, Enterobacter, Pantoea, Paenibacillus, Duganella, Streptomyces, Massilia, Shinella, Janthinobacterium, Erwinia, and Hyphomicrobium were significantly more abundant in the tomato phyllosphere 7 days post-inoculation, suggesting that these rain-borne bacteria successfully colonized the tomato phyllosphere and had a direct impact on the composition of its microbiome. These results were confirmed by comparing the phyllosphere microbiota of tomato plants grown under greenhouse conditions, and thus never exposed to rain, compared to plants grown outside under environmental conditions, including precipitation. Since a large diversity of bacteria is associated with rain, I also hypothesized that rain-borne bacteria are well adapted to environmental stresses, similar to the stressors microbial biopesticides are exposed to in the field. I thus explored rain as a source of resilient biopesticides to control fire blight, caused by the bacterial pathogen Erwinia amylovora, on apple. In an in-vitro dual culture assay, I identified rain-borne isolates displaying broad-range inhibition against E. amylovora and several other plant pathogens. Two rain-borne isolates, identified as Pantoea agglomerans and P. ananatis, showed the strongest inhibition of E. amylovora. Further experiments showed that these two Pantoea isolates survive under environmental conditions and have a strong protective effect against E. amylovora. However, protection from disease in an orchard was inconsistent, suggesting that the timing of application and formulations must be improved for field applications. Using a UV-mutagenesis screen and whole-genome sequencing, I found that a phenazine antibiotic produced by the P. agglomerans isolate was the likely active molecule that inhibited E. amylovora. Bacterial communities are constantly released as aerosols into the atmosphere from plant, soil, and aquatic sources. When in the atmosphere, bacteria may play crucial roles in geochemical processes, including the formation of precipitation. To understand the potential role of decaying vegetation as a source of atmospheric Ice Nucleation Particles (INPs), I analyzed a historic leaf litter sample collected in 1970 that had maintained Ice Nucleation Activity (INA) for 48 years. A culture-dependent analysis identified the bacterial species Pantoea ananatis and the fungal species Mortierella alpina to have INA and to be present in the leaf litter sample. Further, I determined that both P. ananatis and M. alpina produced heat-sensitive sub-micron INPs that may contribute to atmospheric INPs. The development of new sequencing technologies has facilitated our understanding of microbial community composition, assembly, and function. Most research in bacterial community composition is based on the sequencing of a single region of the 16S rRNA gene. Here, I tested the potential of culture-independent 16S rRNA sequencing of the phyllosphere microbiome for disease diagnosis. I compared the community composition of the microbiome of the aerial parts of cheddar pinks (Dianthus gratianopolitanus) that showed disease symptoms with the microbiome of healthy plants to identify the causative agent. However, I found that the pathogen is probably ubiquitous on cheddar pinks since it was present at similar abundance levels in symptomatic as well as healthy plants. Moreover, the low-resolution of 16S rRNA sequencing did not allow to identify the pathogen at the species or strain level. In summary, in this thesis, I found support for the hypothesis that rain is one of the sources of the phyllosphere microbiome, that rain is a promising source of biopesticides to control plant diseases in the field, that leaf litter is a source of atmospheric INPs, and that 16S rRNA sequencing is not well suited for pathogen identification in support of plant disease diagnosis. Finally, in additional research to which I contributed but that is not included in this thesis, I found that metagenomic sequencing can identify pathogens at the species and strain level and can overcome the limitations of 16S rRNA sequencing.
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    Impact of Composting on Drug Residues in Large Animal Mortality
    Ciamillo, Sarah; Peck, Greg; Williams, Mark A.; Splan, Rebecca K.; Porr, C. A. Shea (Virginia Cooperative Extension, 2014-09-25)
    Common methods of mortality disposal include burial, rendering, incineration, and use of a landfill. The availability of options for disposing of mortality, particularly rendering, have changed in recent years, and financially and environmentally sound alternatives are needed.